1. Field of the Invention
The invention relates to genetic factors associated with sensitivity to chemotherapeutic drugs. More particularly, the invention relates to methods for identifying such factors as well as to uses for such factors. The invention specifically provides genetic suppressor elements derived from genes associated with sensitivity of mammalian cells to platinum-based chemotherapeutic drugs, such as cisplatin, and therapeutic and diagnostic uses related thereto.
2. Summary of the Related Art
A broad variety of chemotherapeutic agents are used in the treatment of human cancer. For example the textbook CANCER: Principles & Practice of Oncology, 2d Edition, (De Vita et al., eds.), J. B. Lippincott Company, Philadelphia, Pa. (1985) discloses as major antineoplastic agents the plant alkaloids vincristine, vinblastine, and vindesine; the antibiotics actinomycin-D, doxorubicin, daunorubicin, mithramycin, mitomycin C and bleomycin; the antimetabolites methotrexate, 5-fluorouracil, 5-fluorodeoxyuridine, 6-mercaptopurine, 6-thioguanine, cytosine arabinoside, 5-aza-cytidine and hydroxyurea; the alkylating agents cyclophosphamide, melphalan, busulfan, CCNU, MeCCNU, BCNU, streptozotocin, chlorambucil, bis-diaminedichloroplatinum, azetidinylbenzoquinone; and the miscellaneous agents dacarbazine, mAMSA and mitoxantrone.
Chemotherapeutic agents have proven to be very useful in the treatment of cancer. Unfortunately, some tumor cells become resistant to specific chemotherapeutic agents, in some instances even to multiple chemotherapeutic agents. Such drug resistance or multiple drug resistance can theoretically arise from either the presence of genetic factors that confer resistance to the drugs, or from the absence of genetic factors that confer sensitivity to the drugs. The former type of factors have been identified, and include the multiple drug resistance gene mdr-1 (see Chen et al., 1986, Cell 47: 381-389). However, the latter type of factor remains largely unknown, perhaps in part because the absence of such factors would tend to be a recessive trait.
The platinum coordination complexes, typified by cisplatin (cis-diamminedichloroplatinum (II)) (Reed, 1993, in Cancer, Principles and Practice of Oncology, ibid., pp. 390-4001), have been described as "the most important group of agents now in use for cancer treatment". These agents, used as a part of combination chemotherapy regimens, have been shown to be curative for testicular and ovarian cancers and beneficial for the treatment of lung, bladder and head and neck cancers. DNA damage is believed to be the major determinant of cisplatin cytotoxicity, though this drug also induces other types of cellular damage.
In addition to cisplatin, this group of drugs includes carboplatin, which like cisplatin is used clinically, and other platinum-containing drugs that are under development. These compounds are believed to act by the same or very similar mechanisms, so that conclusions drawn from the study of the bases of cisplatin sensitivity and resistance are expected to be valid for other platinum-containing drugs.
Cisplatin is known to form adducts with DNA and to induce interstrand crosslinks. Adduct formation, through an as yet unknown signalling mechanism, is believed to activate some presently unknown cellular enzymes involved in programmed cell death (apoptosis), the process which is believed to be ultimately responsible for cisplatin cytotoxicity (see Eastman, 1990, Cancer Cells 2: 275-2802).
Many current attempts to optimize the clinical efficacy of platinum compounds are directed at mechanisms that determine cellular sensitivity or resistance to these drugs. Selection of cisplatin-resistant mutant cell lines has revealed several potential mechanisms of cisplatin resistance, none of which have yet been unambiguously proven by molecular genetic analysis (reviewed in Reed, 1993, ibid.).
The first such mechanism involves decreased cellular accumulation of the drug. Observed changes in cisplatin transport in different cell lines may be related to the altered expression of specific membrane proteins. One example of such a protein is a protein termed SQML that is decreased in some cisplatin-resistant cell lines (Bemal et al., 1990, Mol. Cell. Biochem. 95: 61-703). Another such protein is a 200 kilodalton (kDa) glycoprotein which expression has been found to be increased in another such cell line having diminished intracellular accumulation of cisplatin (Kawai et al., 1990, J. Biol. Chem. 265: 13137-13142). Interestingly, a resistance-associated defect in cisplatin uptake has been reported to be a recessive genetic trait, i.e., the phenotype of decreased cisplatin uptake is associated with a loss of gene function (Richon et al., 1987, Cancer Res. 47: 2056-2061).
Another possible mechanism for cisplatin resistance is cytosolic inactivation (termed "quenching") of the drug by sulfhydryl-containing proteins or glutathione. For example, some cisplatin-resistant cell lines have been found to exhibit increased expression of the sulfhydryl-containing proteins, the metallothioneins (Reed, ibid.). Increased levels of glutathione or increased rates of glutathione synthesis have also been correlated with cisplatin resistance (Reed, ibid.). Although it has been reported that transfection of a metallothionein gene into a cell induces cisplatin resistance in such cells (Kelley et al., 1988, Science 241: 1813-1815), these observations have not been confirmed by others (Schilder et al., 1990, Int. J. Cancer 45: 416-422). In another report (Miyazaki et al., 1990, Biochem. Biophys. Res. Commun. 166: 1358-1364) that has not been successfully reproduced by others (Nakagawa et al., 1990, J. Biol. Chem. 265: 4296-4301), cisplatin resistance in cells transfected with the human glutathione-S-transferase (GST)-.pi. gene was correlated with GST-.pi. expression. Furthermore, transfection of cDNAs encoding GSTs of the .mu. and .alpha. classes also has been reported to have had no effect on cisplatin resistance in recipient cells (Leyland-Jones et al., 1991, Cancer Res. 51: 587-594; Townsend et al., 1992, Mol. Pharmiacol. 41: 230-236).
Yet another proposed mechanism to explain cisplatin resistance involves increased repair of DNA adducts. Treatment of cells with non-specific agents that inhibit DNA repair has been reported to sensitize such cells to cisplatin; conversely, increased rates of repair have been observed in some cisplatin-resistant cell lines (Reed, ibid.). It is possible that cisplatin-induced DNA lesions may be repaired by a specialized enzymatic system. Several DNA-binding proteins have been shown to selectively bind to cisplatin-damaged DNA, suggesting that they may be involved in damage repair (Chu & Chang, 1990, Proc. Natl Acad. Sci. USA 87: 3324-3328; Chao et al., 1991, Mol. Cell. Biol. 11: 2075-2080; Bruhn et al., 1992, Proc. Natl. Acad. Sci. USA 89: 2307-2311); however, other functions for these proteins are also conceivable. It has also been reported that transfer of a human DNA repair gene (ERCC-1) to repair-deficient CHO cells led to increased resistance to cisplatin (Reed et al., 1989, Proc. Amer. Assoc. Cancer Res. 30: 488). Paradoxically, when wild-type CHO cells were transfected with the same gene, their cisplatin resistance was reported to decrease rather than increase (Bramson & Panasci, 1993, Cancer Res. 53: 3237-3240), casting doubt on the significance of the earlier observation.
It has also been suggested that pleiotropic regulatory changes induced by the oncogenes H-ras (Sklar, 1988, Cancer Res. 48: 793-797; Isonishi et al., 1991, Cancer Res. 51: 5903-5909; Peters et al., 1993, Int. J. Cancer 54: 450-455), myc (Niimi et al., 1991, Br. J. Cancer 63: 237-241), trk (Peters et al., 1993, ibid.) and fos (Scanlon et al., 1991, Proc. Natl. Acad. Sci. USA 88: 10591-10595) lead to increased cisplatin resistance. Studies with a ribozyme that targets c-fos has suggested that inhibition of c-fos expression may be correlated with cell sensitization to cisplatin (Scanlon et al., ibid.). The association of H-ras with cisplatin resistance, on the other hand, has been called into question by the results of a recent gene transfer study (Perez et al., 1993, Cancer Res. 53: 3771-3775).
Thus, the available evidence suggests the existence of multiple biochemical and physiological mechanisms that are involved in determining cellular sensitivity or resistance to cisplatin. Although several candidate genes responsible for these mechanisms have been suggested, what role, if any, these genes play in cisplatin resistance has not been unambiguously established at the present time.
The unambiguous identification of genes associated with cisplatin sensitivity is thus desirable, because the discovery of such genes can lead to both diagnostic and therapeutic approaches for cancer cells and for drug resistant cancer cells, as well as to improvements in gene therapy and rational drug design. Recently, some developments have been made in the difficult area of isolating recessive genes, including those involved in cytotoxic drug sensitivity. Roninson et al., U.S. Pat. No. 5,217,889 (issued Jun. 8, 1993) teach a generalized method for obtaining genetic suppressor elements (GSEs), which are dominant negative factors that confer the recessive-type phenotype for the gene to which the particular GSE corresponds. (See also Holzmayer et al., 1992, Nucleic Acids Res. 20: 711-717). Gudkov et al., 1993, Proc. Natl. Acad. Sci. USA 90: 3231-3235 teach isolation of GSEs from topoisomerase II cDNA that induce resistance to topoisomerase II-interactive drugs. Abandoned U.S. patent applications Ser. No. 08/033,086, filed Mar. 3, 1993, and Ser. No. 08/177,571, filed Jan. 5, 1994, disclosed the discovery by the present inventors of the novel and unexpected result that GSEs isolated from RNA of cells resistant to the anticancer DNA damaging agent, etoposide, include a GSE encoding an antisense RNA homologous to a portion of a kinesin heavy chain gene. Additionally, abandoned U.S. patent application Ser. No. 08/033,086 disclosed two other GSEs from previously-unknown genes, the expression of said GSEs conferring etoposide resistance on mammalian cells. These results further underscored the power of the GSE technology developed by these inventors to elucidate unexpected mechanisms of drug resistance in cancer cells, thereby providing the opportunity and the means for overcoming drug resistance in cancer patients. This technology has now been applied to isolating and identifying GSEs that confer resistance to cisplatin in cells expressing such GSEs, and for isolating and identifying genes associated with sensitivity of human tumor cells to cisplatin.